The fluorescence of tryptophan (Trp) enables provides a sensitive tool for monitoring ligand binding to proteins. Trp fluorescence is excited with wave lengths of about 280 nm and emission is detected at around 350 nm. The fluorescence intensity depends on solvent exposure. Ligands containing aromatic rings are capable of quenching the fluorescence, and the effect can be used to monitor ligand binding [1]. As proteins usually contain more than a single Trp residue, it is difficult, however, to conclude from the Trp fluorescence on solvent exposure of individual Trp residues or proximity to ligands. Azatryptophans (AzW) are isoelectronic analogues of Trp, and their optical properties show greater sensitivity towards solvent exposure. For example, the fluorescence of 4-AzW and 6-AzW is shifted to longer wavelengths compared to Trp (4-AzW: λ_Excitation = 278 nm; λ_Emission = 423 nm. 6-AzW: λ_Excitation = 325 nm; λ_Emission = 402 nm) [2,3]. Therefore, AzW fluorescence can be detected selectively in the presence of Trp residues. Our experiments showed that the fluorescence intensity of 6-AzW decreases and that of 4-AzW increases when switching from a polar to a non-polar environment. Therefore, site-specific information could be gained, if 4-AzW and 6-AzW can be site-specifically installed in proteins. If corresponding aminoacyl-tRNA synthetases can be identified to genetically encode 4-AzW or 6-AzW, this would be a potent tool to determine the site of ligand binding and investigate the folding rates of individual domains in multi-domain proteins. Preliminary results towards this goal will be presented.